Update on Antimicrobial Research Prepared for Florida Citrus

Update on Antimicrobial Research Prepared for Florida Citrus Industry Annual Conference Bonita Springs, FL June 12, 2014

Requirements for Management • Slow spread of disease – CHMA and insect control • Treat existing infected trees • Protect new plantings • Provide long-term sustainable genetic and biological solutions 11/27/2020 www. citrusrdf. org 2

Requirements for Management • ✔ Slow spread of disease – CHMA and psyllid control – Stewardship of existing products – Label expansions – Bee health - coordination – New insect control actives 11/27/2020 www. citrusrdf. org 3

Requirements for Management • Treat existing infected trees – Enhanced nutritionals – Plant growth regulators – Naturally occurring microbes – Thermal therapy – Antibacterial compounds 11/27/2020 www. citrusrdf. org 4

Outline • Perspective – science, business, regulatory, market aspects of current challenge • Definitions – different languages • Resources available today • What have we learned? • Pipeline of bactericides • From where will the next products come? 11/27/2020 www. citrusrdf. org 5

Some Definitions • Antibacterial treatments • Bactericides and Pesticides – Biopesticides (Microbial and Biochemical) – Antibiotics • Use pattern, residues, tolerance or exemption • Science – evidence over opinions, assumptions explicit, bounds what is known as well as what is unknown, creates technology options 11/27/2020 www. citrusrdf. org 6

Probability Time to Market - Present Investment Vector Pathogen Host 3 YR 6 YR 9 YR Time 11/27/2020 www. citrusrdf. org 7

Bactericides for Crops • To our knowledge in all of US there are only three bactericides approved for use on food crops (excluding copper); Oxytetracycline, Streptomycin and Kasugamycin • This problem is much bigger than citrus but it is ours to solve 11/27/2020 www. citrusrdf. org 8

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Registration of a New Active • A partial list of studies required by EPA (Additional agencies may include USDA-APHIS, FDA, CDC) – (GLN 835. 1240) Soil Column Leaching and (GLN 835. 2410) Photodegradation of Parent and Degradates in Soil, (GLN 835. 6100) Terrestrial Field Dissipation – (GLN 835. 4200) Anaerobic Soil Metabolism, (GLN 835. 4100) Aerobic Soil Metabolism, (GLN 835. 4300) Aerobic Aquatic Metabolism and (GLN 835. 4400) Anaerobic Aquatic Metabolism – (GLN 835. 2120) Hydrolysis of Parent and Degradates as a Function of p. H at 25° C and (GLN 835. 2240) Direct Photolysis Rate of Parent and Degradates in Water 11/27/2020 www. citrusrdf. org 10

Registration of a New Active • Some more EPA data requirements – (GLN 850. 1025) Oyster Acute Toxicity, (GLN 850. 1035) Mysid Acute Toxicity, (GLN 850. 1075) Saltwater Fish Acute Toxicity, (GLN 850. 1075) Freshwater Fish Acute Toxicity, (GLN 850. 1300) Freshwater Invertebrate Life Cycle, (GLN 850. 2300) Avian Reproduction, (GLN 850. 1010) Freshwater Invertebrates Acute Toxicity – (GLN 850. 4100) Terrestrial Plant Toxicity – Tier 1 Seedling Emergence, (GLN 850. 4150) Terrestrial Plant Toxicity – Tier 1 Vegetative Vigor, (GLN 850. 4400) Aquatic Plant Toxicity – Tier 1 Vascular, (GLN 850. 5400) Aquatic Plant Toxicity – Tier 1 Nonvascular 11/27/2020 www. citrusrdf. org 11

Registration of a New Active • Further animal EPA data requirements – (GLN 850. 2100) Avian Oral Toxicity, (GLN 850. 2200) Avian Dietary Toxicity – (GLN 850. 3020) Honeybee Acute Contact Toxicity • Human health – (GLN 870. 7800) Immunotoxicity – Resistance studies with human pathogens planned for frequency of resistance determination • Other studies as directed… 11/27/2020 www. citrusrdf. org 12

Citrus Greening Disease (Huonglongbing) SEARCH FOR AGRICULUTRAL THERAPEUTICS 11/27/2020 13

Biological Assays • Graft-based assay – Infected scion soaked in test solution and grafted onto uninfected rootstock, follow by PCR – Slow, low-throughput – Evidence of efficacy in planta and first look at phyto-toxicity – Open contest with Inno. Centive™ promotion • Liberibacter crescens culture-based assay – Much faster, higher-throughput followed by in planta confirmation on CLas 11/27/2020 www. citrusrdf. org 14

Prior Results • Compounds screened – ~100+ by graft assay and – ~400+ by culture assay • Wide variety of categories of chemicals – Antibiotics and agricultural antibiotics – Polycation polymers – Biopesticides, plant essential oils, terpenoids – New actives and non-antibiotic derivatives – Host immune modulators 11/27/2020 www. citrusrdf. org 15

What are we looking for? • HLB treatment; effects on tree symptoms through CLas titre reduction • Sources: Solutions Page, Research, Companies • Chemical properties – MW: – Log Kow: – p. Ka: < 450 g/mol preferably < 250 g/mol 2 to 4 2 to 6. 5 preferably 3 to 5. 5 • Volume of phloem: 10 ft tree, 1000 L • ? p. H: 8. 0 – 6. 0 stylectomy vs fractionation 11/27/2020 www. citrusrdf. org 16

Relationships • Looking inside the box… – Companies, facilitate turn-key screening • Looking outside the box… – Repurpose products with regulatory advantage – Failed antibiotics with good safety profiles – 25(b) minimal risk pesticides – All commercial compounds fitting our chemical profile and having existing tolerances on other food crops 11/27/2020 www. citrusrdf. org 17

Current Testing Resources Liquid Culture Assay on L. crescens ➪ MIC 90 (Dr. Erik Triplett Laboratory) Citrus Flush Assay on C. asiaticus ➪ Confirmation (Dr. Claudio Gonzalez) Trunk Injection ➪ Dose (Dr. Nian Wang) 11/27/2020 www. citrusrdf. org 18

Requirements and Results Liquid Culture Assay on L. crescens ➪ MIC 90 < 1 mg of compound, ~ 2 weeks for results Citrus Flush Assay on C. asiaticus ➪ Confirmation ~ 100 mg of material, ~ 1 month Trunk Injection ➪ Dose > 10 gr of material, 2 -7 months 11/27/2020 www. citrusrdf. org 19

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Risk Remaining at this Stage • Technical – Commercial scale delivery, efficacy, phytotoxicity • • Cost Regulatory Commercial Partner(s) Market acceptance 11/27/2020 www. citrusrdf. org 21

Build on Success–Expanding Teams • Pivot our focus – connecting corporations • Sponsored research base from CRDF and others, researchers, reviewers, public solutions • Improved communication; knowledge and data sharing between and within grower, researcher, government, corporate sectors • CRDF Board and Committees; RMC, CPDC, IRCC • Research pipeline now enhanced with government funding $21 mm MAC, $25 mm SCRI 11/27/2020 www. citrusrdf. org 22

What comes next? 1. Field trials with top priority Category of Compounds Description Partner Antibiotics Oxytetracycline Streptomycin (Kasugamycin? ) Commercial GRAS-like Plant Essential Oils Natural Products – cymene, carvacrol, etc. CRDF-sponsored formulation research 2. Field trials with major challenges Category of Compounds Description Partner Agricultural Antibiotics Zhongshengmycin Validoxylamine A Source? - Research 11/27/2020 www. citrusrdf. org 23

3. Field trials with commercial partners Category of Compounds Description Partner Biopesticides Surfactin (lipopetides) Company C Host Immune Modifiers One marketed compound One pipeline compound Company C 4. Development pipeline Category of Compounds Description Partner Polycation polymers Proprietary biodegradable polymers Company D Bacterial protein target Ldt. R Lead refinement -Highly active, no human homolog, transcription regulator, essential role in cell wall remodeling Company D (Wang) Lead refinement -Highly active, no human homolog, protein secretion, essential role in effector secretion Non-antibiotic derivatives Tetracycline derivatives Company E (Gonzalez) Bacterial protein target Sec. A 11/27/2020 www. citrusrdf. org 24

Acknowledgements: Bob Shatters, USDA-Ft. Pierce, FL Ed Stover, USDA-Ft. Pierce, FL Chuck Powell, UF-Ft. Pierce, FL Ping Duan, USDA-Ft. Pierce, FL Muqing Zhang, UF-Ft. Pierce, FL Eric Triplett, UF-Gainesville, FL Brij Moudgil, UF-Gainesville, FL Parvesh Sharma, UF-Gainesville, FL Nian Wang, UF-Lake Alfred, FL Claudio Gonzalez, UF-Gainesville, FL Mark Nelson, Echelon Corp. Jim Dukowitz, Technology Innovation Group Harold Browning, Jim Syvertsen, CRDF 11/27/2020 25

Contact: catp@citrusrdf. org solutions@citrusrdf. org tom. turpen@innovationmatters. com THANK-YOU 11/27/2020 26